Dissertation
Dissertation > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)

Hepatitis C virus E2 protein induced cellular transmembrane signal transduction abnormalities and pathogenicity

Author ZhaoLanJuan
Tutor QiZhongTian
School Second Military Medical University
Course Microbiology
Keywords Cellular transmembrane signal transduction E2 protein Pathogenicity Hepatitis virus Phosphorylation Daudi cells Hepatitis Cell Biology behavior HCV infection HepG2 cells
CLC R373
Type PhD thesis
Year 2002
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Currently, the cellular signal transduction abnormalities associated with human disease is one of the hot areas of life science research. More and more evidence of disease and abnormal cell signal transduction is closely related to viruses pathogenic viral antigen may be due to a host that is derived from the intracellular signal transduction of the disorder, which through protein - protein interactions and the regulation of intracellular signaling biological activity of the molecule, thereby changing the characteristics of the normal growth of the host cell. Hepatitis C virus (HCV) infection is a serious liver disease caused by the human major cause. According to statistics HCV infection accounted for 3% of the world's total population, of which more than 50% to chronic persistent hepatitis, some patients may develop cirrhosis or hepatocellular carcinoma, and the lack of satisfactory treatment options, thus greatly endanger human health. Because so far the lack of suitable systems and HCV in vitro proliferation of suitable small animal models, resulting in HCV pathogenesis has not been fully elucidated, and prevention and treatment of HCV infection has become an important issue to be solved field. In addition to liver disease known outside, HCV can also cause a few lymphocytes characterized by abnormal proliferation of diseases such as type II cryoglobulinemia and non-Hodgkin's lymphoma and chronic HCV infection is often accompanied by a variety of extrahepatic itself immune phenomena prompted during HCV infection T, B lymphocyte activation. Why HCV infection led to a different cell (hepatocytes, lymphocytes) lesions occur in the form of the disease characterized? We speculate that this due to HCV of different types of intracellular signal transduction pathways affected differently due. HCV entry into host cells, although the exact mechanisms are not yet very clear, and there are some controversial, but is generally believed that human CD81 protein of HCV E2 specific cell surface receptors, the combination of the way the virus-infected cells. As a class of cellular signal transduction molecules, CD81 involve a very wide range of cell biology behavior; HCV E2 protein with human CD81 interactions not only regulate cell function, viral protein processing and virion formation is also important. Due to HCV and the corresponding target cell surface receptor binding process of starting their pathogenic events in HCV pathogenesis will therefore account for a very important position. At present, all levels from the cellular signal transduction explore HCV pathogenesis studies are concerned intracellular signal transduction changes, the domestic and foreign no HCV E2 protein by specific receptors on the surface of host cells triggered by the target cell transmembrane signaling Transduction pathogenic relationship with HCV reported. The topics to Chinese hamster ovary (CHO) cells expressing recombinant HCV E2 protein, human CD81, MAPK pathway, cell biology acts as a research system, select differentially expressed in human CD81 hepatoma cells, T, B lymphoma cells and tissue lymphoma cells for the study, intended to clarify the HCV E2 protein and the role of human CD81 characteristics; explore the HCV E2 protein by the host cell surface CD81 receptors on intracellular MAPK / ERK and p38MAPK signal transduction pathways, cell biological behavior and its relationship with diseases associated with HCV infection. lt; WP = 7 gt; one, HCV E2 proteins on the MAPK / ERK pathway in flow cytometry analysis showed that, U937 cells do not express human CD81, CD81 expression in the remaining cells in descending order of: Huh-7 cells ? HepG2 cells? Molt-4 cells? Daudi cells. MAPK / ERK kinase activation showed the first 202 threonine and 204 tyrosine (Thr202/Tyr204) phosphorylation reaction occurs, the strength of the phosphorylation reaction reflects the degree of activation of kinases. To determine the HCV E2 protein-activated cell MAPK / ERK kinase suitable conditions, various concentrations of HCV E2 protein in different time stimulate U937, Molt-4, and HepG2 cells. Immunoblotting found that each type of cells in each of the samples of non dependent phosphorylation of MAPK / ERK basically the same amount, to ensure the comparability of the experimental system; 1 mg / L HCV E2 protein is activated cell stimulation 15 min MAPK / ERK suitable conditions. Immunofluorescence U937, Molt-4 and HepG2 cells MAPK / ERK phosphorylation reaction dynamics, three types of immunofluorescence reaction from strong to weak order are: 15 min? 5 min? 30 min? 0 min. Immunohistochemical detection of cell MAPK / ERK phosphorylation reaction: Daudi cells negative reaction; U937 cells weakly positive reaction; Molt-4, Huh-7 cells positive reaction; HepG2 cell response was strongly positive. Observed HCV E2 monoclonal antibody, CD81 antibody, MAPK pathway upstream kinases MEK1 inhibitor-PD98059 on cell MAPK / ERK kinase activation, and found that HCV E2 monoclonal antibody significantly reduced U937, Molt-4 and Huh-7 cells MAPK / ERK activation, inhibition of intracellular L-02 kinase activation. HCV E2 protein CD81 antibody prevents the L-02 cells MAPK / ERK activation was inhibited Huh-7 cells MAPK / ERK activation, decreased Molt-4 cells MAPK / ERK activation, U937 cells from the effects . In addition, HCV infection in the presence of serum HCV E2 antibodies, it can inhibit HCV E2 proteins on the MAPK / ERK activation, inhibition of kinase activation greatest serum dilution due to cell type varies. PD98059 also be different degrees of inhibition of intracellular MAPK / ERK activation. These results indicate that, CHO cells expressing HCV E2 protein having biological activity, can be used as the target cell interactions HCV research tool; signal transduction pathways activated with the signal strength, the duration of the; HCV E2 protein on Daudi cells MAPK / ERK kinase had no effect, but the specific activation of U937, Molt-4, HepG2, Huh-7 and L-02 cells MAPK / ERK kinase activation in different cell lines there are differences between; among other coreceptor outside, HCV E2 mainly by human CD81 receptor protein-activated cell MAPK / ERK kinase; hepatoma cell lines HCV E2 mAb or CD81 mAb sensitive role. Two, HCV E2 proteins on the p38 MAPK pathway of p38 MAPK kinase No. 180 threonine 182 tyrosine (Thr180/Tyr182) phosphorylation reaction occurs that the performance of its activation. Western blotting confirmed, 1 mg / L HCV E2 protein stimulates p38 MAPK kinase 15 min is also suitable activation conditions. Immunohistochemical staining, HCV E2 protein stimulated HepG2 and lt; WP = 8 gt; Huh-7 cells were strongly positive reaction, Molt-4 cells were positive, while the U937 and Daudi cells negative reaction. HCV E2 monoclonal antibody treatment, Huh-7 and L-02 cells p38 MAPK phosphorylation reaction was inhibited; Molt-4 cells was significantly reduced kinase phosphorylation; U937 and Daudi cells p38 MAPK unchanged. HCV-infected patients to prevent HCV E2 proteins on the activation of p38 MAPK, because the degree of inhibition of cell types vary. Human CD81 monoclonal antibody attenuated HCV E2 protein on Molt-4 cells p

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